Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells

Compartmental analysis of ³⁵SO₄²⁻ exchange kinetics is used to obtain SO₄²⁻ fluxes and compartment contents in carrot (Daucus carota L.) storage root cells, where 2 to 5% of the SO₄²⁻ taken up is reduced to organic form. The necessary curve fitting is verified by (a) consistency between `content versus time' and `rate versus time' plots of washout data; (b) agreement between loading and washout kinetics; and (c) correct identification of the fastest exchange phase as being from extracellular spaces.

Sulfate is actively transported up an electrochemical potential gradient at both plasmalemma and tonoplast. The plasmalemma influx is from 2 to 10 times higher than the tonoplast influx, is much greater than the SO₄²⁻ reduction rate, and would not limit the rate of either. This is consistent with the finding that the plasmalemma influx is not regulated by internal SO₄²⁻ or cysteine (Cram 1982 Plant Sci Lett, in press).

Both SO₄²⁻ influxes rise with only limited saturation as the external SO₄²⁻ concentration increases up to 50 millimolarity. Both effluxes appear to be passive, with extensive recycling in the plasmalemma influx pump. SO₄²⁻ permeability is about 10⁻¹¹ meter per second at both membranes.

The high, nonlimiting fluxes of SO₄²⁻ at the plasmalemma relative to the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier 1981 Biochim Biophys Acta 644: 24-35) contrasts with SO₄²⁻ fluxes in bacteria and with Cl⁻ fluxes in plant cells. Their implications for work on characteristics and regulation of SO₄²⁻ uptake in roots and tissue cultures are discussed.

     

Characterization of the nitrate transporter in root plasma membranes of Cucumis sativus L

Nitrate uptake in right-side out plasma membrane vesicles isolated from cucumber roots was characterized. Nitrate uptake into vesicles was driven by an artificially imposed pH gradient. The uptake was strongly inhibited by phenylglyoxal, an arginyl residue modificator. Only a slight repression of NO ₃ ⁻ transport in vesicles was observed in the presence of NEM, a thiol group reagent. pCMBS, an other thiol reagent and DEPC, an effector of histidine residue, had no effect on the nitrate transport in plasma membranes. ATP-driven proton transport in vesicles was not significantly affected in the presence of both, phenylglyoxal and DEPC, whereas pCMBS and NEM abolished it almost completely. The possible role of the particular amino acids residues in the active nitrate transport is discussed. NO ₃ ⁻ uptake into vesicles isolated from both, nitrate-induced and nitrate-depleted plant material was higher than that observed in the vesicles obtained from uninduced plants. Thus, isolated vesicles reflect the well-known in vivo response of intact plants on the exogenous nitrogen regime.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

Influence of cyclopentane beta,beta'-triketones on the ultrastructure of root cap meristematic cells of Cucumis sativus L. seedlings

The influence of cyclopentane beta,beta'-triketones on the ultrastructure of root cap meristematic cells of Cucumis sativus L. under inhibition of root growth by these compounds was studied. It was shown that treatment of the seedlings by these substances at the concentration of 100 microg/ml caused, on the one hand, stimulation of protein-synthesizing apparatus (increase in the nucleolus size and in the number of mitochondria and rough endoplasmic reticulum membranes) and, on the other hand, stimulation of lytic processes. The fact that the used compounds, causing inhibition of the seedling growth, do not suppress but stimulate protein-synthesizing activity of the cells, according to existing concept, testifies to the compound-mediated induction of the cell resistance to unfavourable factors. Consequently, these compounds, in contrast with growth inhibitors suppressing cell metabolism completely, possess properties peculiar to retardants, growth inhibitors valuable for crop production.     

Chilling root temperature causes rapid ultrastructural changes in cortical cells of cucumber (Cucumis sativus L.) root tips

Examination of root tips from cucumber (Cucumis sativus L.) seedlings grown at 8 degrees C for varying periods ranging from 15 min to 96 h, showed marked changes in the ultrastructure of cortical cells within only 15 min of exposure. Greater parts of the cortex were affected with longer periods of exposure, but the sequence of morphological changes in cell components was similar to that found for the roots exposed for 15 min. The effect of chilling injury included alterations in cell walls, nuclei, ER, mitochondria, plastids, and ribosomes. The extent of alterations varied greatly among cells, moderate to severe alterations to cell components being observable among adjoining cells. The measurements of root pressure using the root pressure probe showed a sudden, steep drop in the root pressure in response to lowering of the temperature of the bathing solution from 25 degrees C to 8 degrees C. These observations are discussed in the light of the information available on the ultrastructural and biochemical characteristics of the effect of cold exposure in chilling-sensitive plants.     

A light-enhanced metabolism of sulfite in cells of Cucumis sativus L. cotyledons

The effects of light and several photosynthetic inhibitors on the rate of sulfite metabolism in cells obtained from Cucumis sativus L. cotyledons was studied. The cells were treated with 200 M Na₂SO₃ and the disappearance of sulfite was monitored using either dithiobisnitrobenzoic acid or fuchsin. The rate of sulfite disappearance in light was double the dark rate. Disalicylidene propanediamine at 1 mM increased this light-enhanced metabolism approx. 50%; neither 1 M 3,4-dichlorophenyl-N,N-dimethylurea nor 0.1 mM cyanazine, which completely inhibited CO₂-dependent oxygen evolution, affected the rate of sulfite metabolism. Addition of 200 M Na₂SO₃ to the cells partially inhibited ¹⁴CO₂ fixation. The rate of sulfite consumption by the cells did not affect this inhibition. We conclude that light-dependent sulfite metabolism is cucumber cells may utilize reduced ferredoxin generated as a result of photosynthetic electron transport. An injurious interaction between CO₂ fixation and sulfite appears to occur independently of the sulfite-metabolism process.Abbreviations DCMU 3,4-dichlorophenyl-N,N-dimethylurea - DSPD disalicylidene propanediamine - DTNB 5,5-dithiobis-(2-nitrobenzoic acid)     

Ultrastructure of root cup meristematic cells of Cucumis sativus L. seedlings under inhibition of root growth by synthetic cyclopentene beta,beta'-triketone

A study was made of the ultrastructure of root cap meristematic cells in the seedlings of Cucumis sativus L., whose growth was inhibited by the action of 2-acetyl-4-metylcyclopent-4-en-1,3-dione (AMCD). It is shown that the treatment of seeds by this substance at a concentration of 100 microg/ml leads to the main root inhibition by 73% and, as compared to the control, causes the highest changes in mitochondria that do not reach normal dimensions and undergo degeneration. AMCD caused significant changes in the cell walls that in test samples unusually became electron-dense. Deposits of osmiophylic substance, presumably consisting of terpenoid-like compounds, were often found in the cytoplasm of treated cells as well as in the extracellular space. In many cells, plasmalemma and amiloplast membranes underwent destructive changes. We discuss a possible relation between inhibitory effect of AMCD on the root growth, and its influence on the ultrastructure in investigated cells.     

黄瓜藤的化学成分研究

从丽江产黄瓜藤甲醇提取物的氯仿部位分离得到9个化合物,经理化和波谱分析鉴定为α-菠甾醇(1)、α-菠甾醇-3-O-β-D-葡萄糖苷(2)、β-谷甾醇(β-sitosterol,3)、豆甾-7-烯-3-O-β-D-葡萄糖苷(4)、22-亚甲基-9,19-环羊毛甾烷-3β-醇(5)、(2S,3S,4R,10E)-2-(2′,3′-二羟基二十四烷酰氨基)-10-十八烯-1,3,4-三醇(6)、(2S,3S,4R,10E)-2-[(2′R)-2-羟基二十四烷酰氨基]-10-十八烯-1,3,4-三醇(7)、(2S,3S,4R,10E)-1-(β-D-葡萄糖苷)-2-[(2′R)-2-羟基二十四烷酰氨基]-10-十八烯-1,3,4-三醇(8)、大豆脑苷(9),除化合物3外,其它化合物均为首次从该植物中分离得到.     

外源亚精胺对根际低氧胁迫下黄瓜幼苗光合作用的影响

采用营养液水培法,研究了低氧胁迫下亚精胺(Spd)对黄瓜幼苗净光合速率（P_n）、细胞间隙CO₂浓度（C_i）、气孔导度（G_s）、蒸腾速率（T_r）以及量子效率（Φ_c）和羧化效率（CE）的影响.结果表明,低氧胁迫下,黄瓜植株P_n呈下降趋势,处理10 d后达最低值,为同期对照的63.33%,而低氧胁迫的外源Spd处理10 d时P_n升高了1.25倍;C_i与P_n呈一定负相关性（R²=0.4730～0.7118）,G_s与T_r的变化幅度较大,低氧胁迫下有明显下降趋势,Spd处理后其值有所上升,两者呈显著相关（R²=0.7821～0.9458）,但与P_n的相关性不显著;低氧下Φ_c和CE比对照分别下降了63.01%和72.33%,而Spd处理后,Φ_c和CE值分别提高了23%和14%.表明在低氧胁迫下黄瓜幼苗的光合抑制主要是由非气孔限制所引起的,而外源Spd可通过对光系统的修饰减轻黄瓜幼苗的低氧胁迫伤害.     

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

An interaction is demonstrated between the effects of phytochrome and cryptochrome (the specific blue-light photoreceptor) in the inhibition of hypocotyl elongation of light-grown cucumber (Cucumis sativus L.) cv. Ridge Greenline seedlings. At certain fluence rates of blue light the total inhibition response is greater than the sum of the separate responses to each photoreceptor. The threshold for response to blue light is reduced at least 30-fold by additional red-light irradiation. The synergistic effect is demonstrated for two different fluence rates of red light. Synergism is mediated by phytochrome in both the cotyledons and the hypocotyl.Abbreviations and symbols BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light - photostationary state of phytochrome - _c calculated      

黄瓜果肉色的遗传分析

以1份西双版纳黄瓜和2份普通黄瓜自交系为试验材料配制2个杂交组合,并构建4世代群体(P1、P2、F1、F2).分别采用目测分级、色差仪测定a值和b值以及HPLC方法测定β-胡萝卜素含量等4个指标,研究黄瓜果肉色性状的遗传规律.结果表明:2个杂交组合F2群体的果肉色相关指标呈单峰偏态或双峰分布,表现为主基因+多基因的数量遗传特征.多世代联合分析表明,黄瓜果肉色遗传基本符合两对主基因+多基因模型.两组合8个果肉色相关指标最优模型的F2群体主基因遗传率较高,为76.0%～99.3%,多基因遗传率较低,为0～23.7%.对黄瓜橙黄果肉色的选择应在分离早期世代进行.     

Nitrate transport and compartmentation in cereal root cells

Measurement of cytosolic nitrate is one of the factors requiredfor the resolution of factors controlling nitrate uptake andassimilation in plants and for identifying likely nitrate transportmechanisms at both the plasma membrane and tonoplast. This paperreviews methods and reported measurements of cytosolic nitratein higher plants and concludes that nitrate-selective microelectrodesare the best approach. These microelectrodes have been usedto measure intracellular nitrate activities in barley and maizeroot cells. Triplebarrelled electrodes, incorporating a pH-sensingbarrel have been used to identify the compartmental locationof the nitrate-selective tip giving unequivocal estimates ofvacuolar and cytosolic nitrate activities. The microelectrodemeasurements are used to discuss the possible mechanisms ofnitrate transport at both the tonoplast and plasma membrane.The energetics of possible proton-coupled transport systemsare described and the feasibility of the mechanism is discussed. Key words: Cytosol, compartmentation, Hordeum vulgare L, nitrate, roots, Zea mays L     

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

The control by phytochrome of hypocotyl elongation of light-grown Cucumis sativus L. after a white-light period was examined. The farred-absorbing form of phytochrome inhibits hypocotyl elongation. The response to phytochrome photostationary state () is not linear; all values of from 0.004 to 0.13 promote growth maximally, in the range of values of from 0.13 to 0.22 there is a linear growth response, between values of of 0.22 and 0.35 there is again no differential effect, and for values above 0.35 there is a strong (near linear) effect of on elongation. A kinetic examination of events following the white-light period shows that the major recovery from the photoperiod requires 8.5 h of darkness. End-of-day far-red treatment produces a very different response pattern, with a minor growth stimulation within 28 min of treatment followed by a major effect after 80 to 90 min. Three hours after far-red treatment there is a transient decline in growth rate which persists for about 2 h. Over the whole time course there is a great stimulation of growth rate compared with the controls. A similar growth-rate pattern also occurs if the end-of-day is 0.48, although the magnitude of the growth stimulation is less. Two components are affected by end-of-day , namely the time at which growth recovers and the subsequent growth rate. In the long term, the latter accounts for most of the differences in elongation growth. The dark recovery when only the hypocotyl is irradiated requires 4 h, but end-of-day far-red treatment reduces this to about 1.5 h. The persistence of the far-red-absorbing form of phytochrome for many hours in darkness in these light-grown plants is also demonstrated.Abbreviations and symbols D darkness - FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light - WL white light (from fluorescent lamps) - photostationary state of phytochrome - _c calculated      

Calcium absorption as an index of root parameters of cucumber (Cucumis sativus L.)

Summary In a solution culture study the absorption of Ca in the shoot of cucumber (Cucumis sativus L.) and various root parameters viz fresh weight, dry weight, length and surface area were measured at weekly intervals. All the root parameters were significantly correlated (p=0.01) with the amount of Ca absorbed when the concentration of Ca was such (5 meq/l) that the absorption capacity of the root membrane was fully saturated. The use of this relationship has been suggested in estimating the amount of roots developed in the soil system where identical conditions exist with respect to Ca concentration. Such estimated values have been termed as equivalent root length or equivalent root surface area as the case may be. P and K flux into the roots growing in the soil system have been worked out from these estimates of root parameters.     

钙对黄瓜子叶愈伤组织中硝酸还原酶（NR）活性的影响

本文采用黄瓜叶愈伤组织,研究了不同钙培养下,对愈伤组织生长、硝酸盐吸收,NR活性以及组织中钙、镁含量的影响。结果表明,缺钙后,愈伤组织生长、硝酸盐吸收、NR活性等都比正常钙培养下的愈伤组织降低。这与用整株植物所得结果基本一致,对此结果进行了讨论。     

硝酸银对雌性黄瓜植株三种氧化酶同工酶的影响

雌性黄瓜植株经硝酸银处理后其茎尖和真叶过氧化物酶活性极显著地增加,茎尖24小时、真叶36小时酶活性达到最大值,分别增加了178.2%和284.6%,随后酶活性逐渐下降,但酶活性仍然较对照植株高。多酚氧化酶和超氧化物歧化酶的同工酶活性也增加。同时硝酸银能诱发黄瓜植株过氧化物酶、多酚氧化酶和超氧化物歧化酶产生新的同工酶, 用等电聚焦更能有效地观察新产生的同工酶。     

The control of food mobilization in seeds of Cucumis sativus L.

An analysis of the in vitro activities of proteolytic enzymes from cotyledons of germinating cucumber seeds has been carried out and the effects of protein degradation products on such activities monitored. Aminopeptidase activity is substantially inhibited with either L-leucine or L-phenylalanine and trypsin activity with L-arginine. Aminopeptidase activity was also markedly reduced in the presence of individual di- and tripeptides. Of the peptides tested, however, only L-tryptophyl-L-phenylalanine inhibited the degradation of native cucumber seed protein by the endogenous cucumber seed protease(s) (autodigestive activity).Abbreviations TCA trichloroacetic acid - L-leuglygly L-leucylglycylglycine - L-pheglygly L-phenylalanylglycylglycine - L-phe-L-leu L-phenylalanyl-L-leucine - L-leu-L-phe L-leucyl-L-phenylalanine - L-tryp-L-phe L-tryptophyl-L-phenylalanine - LPA L-leucine-p nitroanilide - BAPNA -N-benzoyl-DL-arginine-p nitroanilide - ADA autodigestive activity